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Ishii, J., Izawa, K., Matsumura, S., Wakamura, K., Tanino, T., Tanaka, T., Ogino, C., Fukuda, H. and Kondo, A. (2009) A simple and immediate method for simultaneously evaluating expression level and plasmid maintenance in yeast. Journal of Biochemistry, 145, 701-708. doi:10.1093/jb/mvp028

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• TITLE: A versatile vector system for generating recombinant EGFP-tagged proteins in yeast

  AUTHORS: Francesco Palma, Laura Chiarantini

  KEYWORDS: Enhanced GFP; Protein Localization; Tuber borchii Vittad; Saccharomyces cerevisiae

  JOURNAL NAME: Advances in Bioscience and Biotechnology, Vol.2 No.1, February 24, 2011

  ABSTRACT: This paper reports a versatile egfp-tagged pFL61-based expression vector system which allows the production on yeast of homo- and heterologous proteins fused with the Enhanced Green Fluorescent Protein (EGFP) at the C-terminus. This expression system, which involves a fluorescent protein, readily allows both to verify the expression and to localize the protein in the yeast cell. The vector carries a Not I site upstream the first codon of the egfp gene. The yeast cells harbouring this plasmid emit a feeble emission compared to the fluorescence expected. Was then investigated the effect of the Not I site, located very close to the start codon, on the expression of the reporter egfp gene using northern and western blotting, fluorescence microscopy and flow cytometry. Data indicated that this palindromic site could hide the start codon so as to negatively affect translation. This aspect confers to the proposed expression system an advantage in distinguishing clones after transformation.